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Ultramicroscopy 109 (2009) 344–349

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Ultramicroscopy

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A ﬂuorescence scanning electron microscope

Takaaki Kanemaru a, Kazuho Hirata b,Ã, Shin-ichi Takasu c, Shin-ichiro Isobe d, Keiji Mizuki e, Shuntaro Mataka f, Kei-ichiro Nakamura g a Morphology and Core Unit, Kyushu University Hospital, Kyushu, Japan b Department of Anatomy and Cell Biology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Maidashi 3-1-1, Fukuoka 812-8582, Japan c Advanced Technology Division, JEOL Ltd., Tokyo, Japan d Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kyushu Sangyo University, Fukuoka, Japan e Department of Nanoscience, Faculty of Engineering, Sojo University, Kumamoto, Japan f International Science Technology Co., Ltd, Kasuga Laboratory, Fukuoka, Japan g Department of Anatomy, Kurume University School of Medicine, Kurume, Japan article info abstract

Article history: Fluorescence techniques are widely used in biological research to examine molecular localization, while Received 12 August 2008 electron microscopy can provide unique ultrastructural information. To date, correlative images from Received in revised form both fluorescence and electron microscopy have been obtained separately using two different 22 December 2008 instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current Accepted 6 January 2009 study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In Keywords: the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were Correlative microscopy employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative Scanning electron microscopy fluorescence and electron microscopy. Fluorescence microscopy & 2009 Elsevier B.V. All rights reserved. Organic EL fluorophore

1. Introduction localization. Fluorescent probes, which label an identical region of a single specimen not only for fluorescence microscopy but also Fluorescence microscopy has become an indispensable micro- for electron microscopy [6], such as FluoroNanogold [7], and more scopy technique for the examination of biological specimens, recently ReAsH, which is used in the tetracystein–biarsenical because it allows selective and specific detection of molecules at system [8] and small nanocrystals (Quantum dots; QDs) [9], have small concentrations with a good signal-to-background ratio [1,2]. all been being utilized; nevertheless, complications during the It even allows one to work with intact samples, including living specimen preparation process and during imaging with both types cells, and to see samples with the naked eye; these advantages are of microscopy are unavoidable. not available with other methods, such as electron microscopy [3]. One of the ways in which the complex methods for correlative Furthermore, recent developments in fluorescence imaging microscopy could be improved would be to incorporate one techniques have enabled the well-known Abbe barrier of about microscope into another. The technology of the scanning electron 200 nm lateral resolution to be crossed, that is, the diffraction microscope (SEM) is now well advanced and the image resolution limit for an optical microscope has approached the level of 100 nm of the SEM approaches that of the transmission electron as with 3D structural illumination microscopy (3D-SIM) [4] microscope (TEM) [10]. It is clear that for the majority of biological or even less than 100 nm, as with other techniques such as 4Pi, samples, it is easier and less time-consuming to prepare samples simulated emission depletion (STED) and photoactivated localiza- for scanning electron microscopy than for transmission electron tion (PALM) [3,5]. microscopy. Furthermore, the structure of the SEM makes it easier In order to obtain a stable image with a higher resolution, and more cost-effective to incorporate other devices into it. This however, an electron microscope is still required. Corre- led us to attempt to make a hybrid ‘‘FL-SEM’’ instrument in which lative study using both fluorescence and electron microscopy is an SEM is combined with an FM. In parallel with its set-up, some usually employed to obtain substantial information on molecular fluorophores were tested for the labeling of biological samples using the new instrument. We found that an organic EL fluorophore named Fluolid (Pub. no. US 7015002) [11], which has a high physical stability was suitable for this purpose. Ã Corresponding author. Tel.: +8192 642 6048; fax: +8192 642 6050. The design of the FL-SEM is described and the first FL-SEM E-mail address: [email protected] (K. Hirata). images are presented.

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Some of our ﬁndings have been previously reported in abstract possible ﬂuorescence damage which may be caused by the form [12]. electron beam. Each of the FM and SEM images is separately displayed on a single PC screen through the CCD camera and the AD converter, respectively (Fig. 1C). Both images are then 2. Experimental manually merged using Adobe Photoshop CS.

2.1. Assembly of the FL-SEM 2.2. Animals

An SEM for wavelength dispersive spectrometry (WDS) (JEOL Under deep anesthesia induced by diethyl ether, adult male JXA8600 M, JEOL, Tokyo Japan) was reconstructed to create a Wistar rats were perfused intracardially with PBS, followed dual-mode microscope, named the FL-SEM, which is capable of by a mixture of 2.8% paraformaldehyde, 0.2% picric acid and obtaining both FM and SEM images without requiring to move the 0.06% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. specimen (Fig. 1A and B). All the parts of the built-in optical The diaphragm and the kidney were removed and postfixed with microscope unit in the SEM for WDS, with the exception of a 4% paraformaldehyde in 0.1 M PB. mirror and the Cassegrain type (non-chromatic aberration type) These experiments were reviewed by the Committee on Ethics 45 , NA 0.41 optical objective lens, both of which are placed Â for Animal Experiments of the Faculty of Medicine, Kyushu within the column and have a small hole for the electron beam to University and were carried out according to the guidelines for pass through, were replaced with an FM fitted with a digital Animal Experiments of the University, and Law no. 105 and camera unit that was assembled in our laboratory. The unit Notification no. 6 of the Japanese Government. consisted of a laser light source from an external unit (473 nm, Showa Optronics, Tokyo, Japan) (a in Fig. 1A and B), an emission filter (515 nm LP), an adaptor device for concentrating the laser 2.3. Exploitation of a new probe for FL-SEM light (b in Fig. 1A and B), a unit consisting of mirrors and prisms (c in Fig. 1A and B), an external CCD camera (Bitran Corporation, In a preliminary study, we attempted to use some tissues Saitama, Japan) adapted via a C-mount adaptor (d in Fig. 1A and that had been labeled with GFP or FITC as FL-SEM samples. B), and an eye piece (e in Fig. 1A and B). In the FL-SEM, an electron These fluorophores were almost bleached out in the process of beam passes through small holes at the center of a mirror and the dehydration. For this reason, we employed a new probe, Fluolid, object lens in order to reach the specimen. The SEM image is then which is a small organic fluorophore originally synthesized as an sent to an AD converter (SemAfor, JEOL SA20) via a photomulti- organic EL dye in our laboratory (Pub. no. US 7015002), [11]. The plier (PMT). On the other hand, the excitation beam from the Fluolid dye has a high physical stability and a large Stokes shift external unit reaches the specimen along the same passage as the and it shows strong fluorescence intensity in its solid state. electron beam within the column. The fluorescence emission from However, it has never been applied to the labeling of biological specimens is then directed to an external CCD camera. In practice, samples. In order to test its viability, a kidney was immersed in the digital image from the FM is acquired first, with the image 20% sucrose in PBS and 10-mm-thick frozen sections of the kidney from the SEM being sequentially acquired, in consideration of were made for peanut agglutinin (PNA) staining, in accordance

Fig. 1. (A). A view of the FL-SEM. (B). A schematic diagram of an inside view of the FL-SEM, which is made up of a combination of the SEM and the FM units. (C). A schematic diagram depicting the ﬂow of SEM and FM image signals to a PC display. (a): the laser light source of the external unit (473 nm), (b): an adaptor device for laser light, (c): mirror and prism, (d): external CCD camera, and (e): eye piece. PMT, photomultiplier tube. Author's personal copy ARTICLE IN PRESS

346 T. Kanemaru et al. / Ultramicroscopy 109 (2009) 344–349 with a report by Holthofer [13] regarding specific PNA binding Fluolid dye was able to successfully label the PNA binding site to the brush border of epithelial cells within the renal proximal of the apical region of the renal proximal tubule cells (Fig. 2A). tubules. Fluolid-W-Orange was conjugated with streptavidin The fluorescent intensity of Fluolid became stronger in the same utilizing the IST Fluolid-protein-labeling kit (CosmoBio, Japan) section mounted with non-aqueous Histomount (Invitrogen) after before use. After being washed with PBS, the sections were dehydration (Fig. 2B), whereas that of the FITC was attenuated incubated with biotinylated PNA (Vector) (1:100) at room (Fig. 2C and D). Furthermore, Fluolid showed no change over 4 temperature (RT) overnight and then with Fluolid-W-Orange- months, even under direct light (Fig. 2E and F), whereas the FITC conjugated streptavidin (1:10) at RT for 3 h. The sections were was completely bleached out (Fig. 2G and H). Alexa Fluor, which is then mounted with aqueous Vectashield (Vector). FITC-conjugated known to have a strong photostability [14], showed similar streptavidin (Vector) (1:100) and Alexa-488-conjugated strepta- findings to Fluolid (data not shown). Thus, Fluolid and Alexa vidin (Molecular Probe) (1:500) were used as controls. Under Fluor were confirmed to be suitable for specimen preparation for conventional FM (Zeiss Axiophoto), it was revealed that the the FL-SEM.

Fig. 2. Conventional ﬂuorescent micrographs of the PNA staining of frozen sections of the rat kidney, which was labeled by Fluolid-W-Orange, an organic EL dye. (A, B, E, and F). FITC (C, D, G, and H) was used as a control. The stability of the ﬂuorophores to dehydration (A–D) and daylight (E–H) is shown. Scale bar: (A–D): 20 lm, (E–H): 100 lm. Author's personal copy ARTICLE IN PRESS

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2.4. Preparation of FL-SEM samples The identical region was also recorded through the SEM unit without moving the specimen (Fig. 3B). Both images were finally For FL-SEM specimens, the fixed diaphragm and kidney were merged on the PC display (Fig. 3C). In the merged image, the Iba1- processed for Iba1 immunohistochemistry and PNA staining, positive macrophages distributed among the muscle fibers were respectively. The immunohistochemical procedure used here has clearly indicated (Fig. 3C). Details of the surface structure of one of been described elsewhere [15]. Briefly, after removing the the macrophages were further observed at high resolution of the peritoneum, the diaphragm was preincubated with 1% bovine SEM (8000 )(Fig. 3D). Thus the FL-SEM allows spatial localiza- Â serum albumin in PBS at RT, for 1 h, in order to block nonspecific tion of a cell via its molecular expression and its 3-D character- binding sites. The specimen was then incubated with a rabbit ization at higher magnification. At the same time, this new polyclonal anti-Iba1-antibody (Wako) (1:100) as the primary instrument has also solved the problem of locating the site of antibody at RT for 3 days and with an Alexa488-anti-rabbit interest for SEM observation, through the process of prescreening antibody (Molecular Probes) (1:200) as the secondary antibody at with a fluorophore-labeled structure. RT for 1 day. The labeled specimen was then dehydrated with Next, the bulk of the Fluolid-labeled fractured kidney, in which acetone and coated with osmium (2.5 mm in thickness) in an ion-etching technique was applied to delineate the subcellular an osmium-plasma coater (HPC-1S, Vacuum Device Inc., Ibaragi). structure, was observed (Fig. 4A–B). In the merged image, the The fixed kidney was freeze-fractured using DMSO and was first labeling was detected in the apical region of the epithelial cells incubated with biotinylated PNA (Vector) (1:100) at RT for 3 days within the proximal tubules (Fig. 4A, an arrow). The enlargement and then with Fluolid-W-Orange-conjugated streptavidin (1:10) at of the labeled site by the SEM demonstrated that the Fluolid- RT for 1 day. Some of the labeled specimens were thinly sectioned labeled site almost corresponded to the thick brush border after being embedded with Technovit 8100 resin (Heraeus Kulzer) consisting of tall microvilli that were sharply delineated by the (cf. Fig. 4C and D, about 5 mm thick), due to the limitation of ion-etching (Fig. 4B). In the thick bulk sample, we sometimes the FM with respect to its depth of focus [1], compared to the encountered non-specific fluorescent sites (Fig. 4A, arrowheads). relatively large depth of focus of the SEM [10]. In order to This is probably because the entire sample was excited indis- delineate the subcellular structure, an additional technique of ion- criminately and most of the fluorescent photons arose from out- etching [16,17] was employed. Very mild rapid etching was of-focus fluorophores [1]. Therefore, a thin section of the kidney in conducted as follows: 12 mA for 8 min, employing an ion coater which ion-etching was also carried out was further observed. The above the sample bulk on silicon ware. The specimens were then fluorescent image was dramatically improved; the labeling was immersed in 100% acetone for 140 min and were processed for restricted to the apical region of the epithelial cells (Fig. 4C), osmium-plasma coating. which corresponded to the microvilli (Fig. 4D). Thus, by using a fluorophore-labeled section through the cells, the FL-SEM is also capable of analyzing correlations between the FM and the SEM 3. Results and discussion images of the intracellular structure. The necessity of a dual mode SEM equipped with an optical The bulk of the Alexa Fluor-labeled diaphragm was first microscope was stressed by Yamada et al. [18] who characterized observed by FL-SEM. The image at the maximum resolution of the constituent distribution of food tissues utilizing a color SEM the FM (650 ) was recorded through the FM unit (Fig. 3A). image obtained from two separate instruments through digital Â

Fig. 3. FL-SEM imaging of Iba1-immunostained rat diaphragm. FM (A) and SEM (B) images and the merged image (C) of an identical region. (D) An enlarged SEM image of the white box in (C). Alexa 488-labeled Iba1-positive macrophages (green) can be seen among the skeletal muscles. Scale bar: (A–C): 10 lm, (D): 1 lm. Author's personal copy ARTICLE IN PRESS

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Fig. 4. FL-SEM imaging of the PNA-stained fractured bulk (A and B) and a thin section (C and D) of the rat kidney that have been treated with an ion-etching technique to delineate the subcellular structure. A–B: Merged images (A) and an enlarged SEM image (B) of the Fluolid-W-orange-labeled PNA-positive site (orange) indicated by an arrow in (A). Arrowheads indicate non-specific fluorescent sites. (C–D): The merged image (C) and the SEM image (D) of a cross-section of the renal proximal tubule. Note that the labeling (orange) is precisely superimposed onto the microvilli. Asterisks indicate the nuclei of the corresponding endothelial cells within the proximal tubule. Scale bar: (A): 10 lm, (B): 1 lm, and (C–D): 10 lm. image processing. Boyde et al. [19] analyzed bone tissues with the T. Kondo and Dr. K. Ohta for their helpful discussions. 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This hybrid instrument could SEM with a fluorescence microscope unit, in: Proceedings of the 16th be a practical tool with a high throughput for biological research. International Microscopy Congress, 2006, p. 664. [13] H. Holthofer, Lectin binding sites in kidney. A comparative study of 14 animal species, J. Histochem. Cytochem. 31 (1983) 531–537. [14] R. Haugland, Handbook of Fluorescent Probes and Research Products, ninth Acknowledgements ed., Molecular Probes, Eugene, OR, 2006. [15] K. Yasuoka, K. Hirata, A. Kuoraoka, J. He, M. Kawabuchi, Expression of amyloid precursor protein-like molecule in astroglial cells of the subventricular We thank A. Togo, K. Yamashita, and N. Yamaguchi for their zone and rostral migratory stream of the adult rat forebrain, J Anat 205 (2004) technical support; Dr. F. Ishikawa for helpful suggestions; and Dr. 135–146. Author's personal copy ARTICLE IN PRESS

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